Malone Cheryl L, Boles Blaise R, Lauderdale Katherine J, Thoendel Matthew, Kavanaugh Jeffrey S, Horswill Alexander R
Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
J Microbiol Methods. 2009 Jun;77(3):251-60. doi: 10.1016/j.mimet.2009.02.011. Epub 2009 Mar 3.
With the emergence of Staphylococcus aureus as a prominent pathogen in community and healthcare settings, there is a growing need for effective reporter tools to facilitate physiology and pathogenesis studies. Fluorescent proteins are ideal as reporters for their convenience in monitoring gene expression, performing host interaction studies, and monitoring biofilm growth. We have developed a suite of fluorescent reporter plasmids for labeling S. aureus cells. These plasmids encode either green fluorescent protein (GFP) or higher wavelength reporter variants for yellow (YFP) and red (mCherry) labeling. The reporters were placed under control of characterized promoters to enable constitutive or inducible expression. Additionally, plasmids were assembled with fluorescent reporters under control of the agr quorum-sensing and sigma factor B promoters, and the fluorescent response with wildtype and relevant mutant strains was characterized. Interestingly, reporter expression displayed a strong dependence on ribosome binding site (RBS) sequence, with the superoxide dismutase RBS displaying the strongest expression kinetics of the sequences examined. To test the robustness of the reporter plasmids, cell imaging was performed with fluorescence microscopy and cell populations were separated using florescence-activated cell sorting (FACS), demonstrating the possibilities of simultaneous monitoring of multiple S. aureus properties. Finally, a constitutive YFP reporter displayed stable, robust labeling of biofilm growth in a flow-cell apparatus. This toolbox of fluorescent reporter plasmids will facilitate cell labeling for a variety of different experimental applications.
随着金黄色葡萄球菌在社区和医疗机构中成为一种突出的病原体，对有效的报告工具以促进生理学和发病机制研究的需求日益增长。荧光蛋白作为报告分子非常理想，因为它们便于监测基因表达、进行宿主相互作用研究以及监测生物膜生长。我们开发了一套用于标记金黄色葡萄球菌细胞的荧光报告质粒。这些质粒编码绿色荧光蛋白（GFP）或用于黄色（YFP）和红色（mCherry）标记的更高波长报告变体。报告基因置于已表征启动子的控制之下，以实现组成型或诱导型表达。此外，将质粒与受agr群体感应和σ因子B启动子控制的荧光报告基因组装在一起，并对野生型和相关突变菌株的荧光反应进行了表征。有趣的是，报告基因的表达对核糖体结合位点（RBS）序列有很强的依赖性，在所检测的序列中，超氧化物歧化酶RBS表现出最强的表达动力学。为了测试报告质粒的稳健性，使用荧光显微镜进行细胞成像，并使用荧光激活细胞分选（FACS）分离细胞群体，证明了同时监测多种金黄色葡萄球菌特性的可能性。最后，一个组成型YFP报告基因在流动细胞装置中对生物膜生长表现出稳定、稳健的标记。这套荧光报告质粒工具箱将便于在各种不同的实验应用中进行细胞标记。
